CN115970654A - Efficient synthesis process and application of magnetic microspheres - Google Patents
Efficient synthesis process and application of magnetic microspheres Download PDFInfo
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Abstract
The invention provides an efficient synthesis process of magnetic microspheres and application thereof. The invention firstly dopes the graphene oxide with Fe 3 O 4 The magnetic core is prepared on the basis, and the magnetic microsphere with high-efficiency separation and purification performance is successfully prepared by taking chitosan as the shell. Proved by experiments, the recovery rate of the magnetic microspheres prepared by the invention to nucleic acid can reach 96 percentThe recovery rate of the protein can reach more than 92 percent, and the technical effect which is comparable to that of the magnetic beads sold on the market is achieved, compared with the pure Fe which is not modified at all 3 O 4 As for the nano magnetic microspheres and the chitosan microspheres, the magnetic microspheres prepared by the method are more suitable for purification and separation of nucleic acid and protein, and have very large market application prospects.
Description
Technical Field
The invention relates to the technical field of nucleic acid and protein separation and purification, in particular to a high-efficiency synthesis process of magnetic microspheres and application thereof.
Background
The extraction of nucleic acid, protein and other active substances from biological samples is a basic technology in the field of life science research such as molecular biology and clinical medicine. The traditional method for extracting nucleic acid, such as phenol-chloroform extraction method, has the defects of high toxicity of organic solvent, more repetitive labor, difficulty in realizing miniaturization and automation operation and the like. Protein separation and purification are core technologies in the modern biological industry, and have the problems of high technical difficulty, high cost and the like, and currently, the commonly used protein separation and purification technologies include: precipitation, electrophoresis, dialysis, chromatography, etc., but all of the above techniques have a problem of low efficiency of separation and purification.
The magnetic particle as a novel solid phase extracting agent for separating nucleic acid and protein can further simplify and accelerate the separation and purification process of nucleic acid and protein by means of a proper magnetic separation device, and realize the miniaturization, automation and parallelization of the purification process of nucleic acid and protein. Recently, it has been reported that the novel magnetic silica microspheres have the characteristics of regular spherical shape, narrow particle size distribution and high magnetic responsiveness, and are particularly suitable for extracting nucleic acid in a high-viscosity sample solution. However, the existing magnetic microsphere preparation technologies cannot simultaneously realize quick and efficient bio-separation, and the purification efficiency of detection also faces high-quality requirements.
Disclosure of Invention
Aiming at the current situation, the invention provides an efficient synthesis process and application of magnetic microspheres. Proved by experiments, the invention firstly dopes the graphene oxide with Fe 3 O 4 The magnetic core is prepared on the basis, and the magnetic microsphere with high-efficiency separation and purification performance is successfully prepared by taking chitosan as the shell. Proved by experiments, the recovery rate of the magnetic microspheres prepared by the invention to nucleic acid can reach more than 96 percent, the recovery rate to protein can reach more than 92 percent, and the magnetic microspheres are obtainedCompared with the magnetic beads on the market, the magnetic beads have the technical effect. Compared with pure Fe without any modification 3 O 4 As for the nano magnetic microspheres and the chitosan microspheres, the magnetic microspheres prepared by the method are more suitable for purification and separation of nucleic acid and protein, and have very large market application prospects.
Specifically, the invention provides an efficient synthesis method of magnetic microspheres, which comprises the following steps:
1) Preparing a magnetic core: preparing graphene oxide and ferric chloride by a hydrothermal synthesis method to obtain a magnetic core;
2) Modification of a chitosan shell: modifying a chitosan shell on the surface of the magnetic core synthesized in the step 1) to prepare the magnetic microsphere.
Preferably, the synthesis method of step 1) comprises the following steps: firstly, dissolving graphene oxide in a solution of ethylene glycol and diethylene glycol, then adding sodium acetate and ferric chloride, and mixing uniformly. Then, the mixed solution was transferred to a stainless steel reactor and heated at 200 ℃. After the reaction is finished, washing the reaction product by using ethanol to remove unreacted impurities. And the mixture was transferred to a dialysis bag and dialyzed slowly for two days. Finally, the product was collected by magnetic separation and resuspended in deionized water.
Further preferably, the synthesis method of step 1) comprises the following steps: firstly, 10-30mg of graphene oxide is dissolved in a solution of 0.5-1.5mL of ethylene glycol and 9.5-28.5mL of diethylene glycol, and then 0.2830-0.8490g of sodium acetate and 270-810mg of ferric chloride are added and mixed uniformly. Then, the mixed solution is transferred to a stainless steel reaction kettle and heated for 10 to 30 hours at the temperature of 200 ℃. After the reaction is finished, washing the reaction product by using ethanol to remove unreacted impurities. And transferring the mixed solution into a dialysis bag (MWCO 8000-14000) for slow dialysis for two days. Finally, the product was collected by magnetic separation and resuspended in deionized water.
Preferably, the synthesis method of step 2) comprises the following steps: firstly, chitosan is dissolved in acetic acid solution and is fully dissolved by ultrasonic dispersion. Then taking out the product synthesized in the step 1), adding EDC and NHS at the same time, and mixing uniformly. Under the protection of nitrogen, the reaction is carried out so as to activate the carboxyl on the graphene. Subsequently, the chitosan solution prepared above was added and allowed to react for two days at room temperature. After the reaction, the mixture was transferred to a dialysis bag and dialyzed slowly for two days. And finally, collecting the product through magnetic separation, and suspending the product in deionized water to obtain the magnetic microsphere.
Further preferably, the synthesis method of step 2) comprises the following steps: firstly, 0.1-0.3g of chitosan is dissolved in 14-42mL0.04M acetic acid solution, and the chitosan is fully dissolved by ultrasonic dispersion. Then 7-21mL of the product synthesized in step 1) was taken out, and 1-3mL0.5M EDC and 1-3mL0.96M NHS were added at the same time and mixed well. And under the protection of nitrogen, carrying out the reaction for 4-8 hours so as to activate the carboxyl on the graphene. Subsequently, the chitosan solution prepared above was added and allowed to react for two days at room temperature. After the reaction, the mixture was transferred to a dialysis bag (MWCO 8000-14000) and dialyzed slowly for two days. And finally, collecting the product through magnetic separation, and suspending the product in 50-150mL of deionized water to obtain the magnetic microsphere.
In another aspect, the present invention provides a method for efficiently synthesizing magnetic microspheres, comprising the following steps:
1) Preparing a magnetic core: firstly, 10-30mg of graphene oxide is dissolved in a solution of 0.5-1.5mL of ethylene glycol and 9.5-28.5mL of diethylene glycol, and then 0.2830-0.8490g of sodium acetate and 270-810mg of ferric chloride are added and mixed uniformly. Then, the mixed solution is transferred to a stainless steel reaction kettle and heated for 10 to 30 hours at the temperature of 200 ℃. After the reaction is finished, washing the reaction product by using ethanol to remove unreacted impurities. And transferring the mixed solution into a dialysis bag (MWCO 8000-14000) for slow dialysis for two days. Finally, the product was collected by magnetic separation and resuspended in deionized water.
2) Modification of a chitosan shell: firstly, 0.1-0.3g of chitosan is dissolved in 14-42mL0.04M acetic acid solution, and the chitosan is fully dissolved by ultrasonic dispersion. Then 7-21mL of the product synthesized in step 1 was taken out, and 1-3mL0.5M EDC and 1-3mL0.96M NHS were added at the same time and mixed well. And carrying out reaction for 4-8 hours under the protection of nitrogen so as to activate the carboxyl on the graphene. Subsequently, the chitosan solution prepared above was added and allowed to react for two days at room temperature. After the reaction, the mixture was transferred to a dialysis bag (MWCO 8000-14000) and dialyzed slowly for two days. And finally, collecting the product through magnetic separation, and suspending the product in 50-150mL of deionized water to obtain the magnetic microsphere.
In another aspect, the invention provides an application of the magnetic microsphere in nucleic acid separation and purification.
Further preferably, the application is that the magnetic microspheres prepared by the invention are contacted with a nucleic acid sample to be separated, and the nucleic acid separation and purification can be realized through magnetic separation.
Preferably, the nucleic acid sample to be isolated is genomic DNA of E.coli.
In another aspect, the invention provides an application of the magnetic microsphere in protein separation and purification.
Further preferably, the application is that the magnetic microspheres prepared by the invention are contacted with a protein sample to be separated, and the protein separation and purification can be realized through magnetic separation.
Preferably, the protein sample to be separated is BSA.
The invention has the following advantages:
1) The invention firstly dopes the graphene oxide with Fe 3 O 4 Preparation of magnetic cores on the basis of Fe in comparison with Fe alone 3 O 4 As for the magnetic core constructed on the basis, because the graphene oxide has a monomolecular layer two-dimensional network structure similar to that of graphene and also has oxygen-containing functional groups such as carboxyl, hydroxyl, epoxy and the like, GO has the advantages of excellent shielding performance, high length-diameter ratio, ultrahigh strength, ultrahigh heat conductivity, high surface activity and the like of graphene, and also has excellent physical, chemical, optical and electrical propertiesAnd the GO contains a large number of oxygen-containing active groups, so that the solubility of the GO in a polar solvent is improved, and covalent bond functionalization of graphene is facilitated, so that a foundation is laid for subsequent chitosan shell preparation.
2) Experiments prove that the recovery rate of the magnetic microspheres prepared by the method for nucleic acid can reach more than 96%, the technical effect which is comparable with that of the commercially available AMpureXP magnetic beads is achieved, and meanwhile, the recovery rate of the magnetic microspheres for protein can reach more than 92%. Compared with pure Fe without any modification 3 O 4 As for the nano magnetic microspheres and the chitosan microspheres, the magnetic microspheres prepared by the method are more suitable for purification and separation of nucleic acid and protein, and have very large market application prospects.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all the technical means used are conventional means well known to those skilled in the art.
Example 1
The invention provides a high-efficiency synthesis method of magnetic microspheres, which comprises the following steps:
1) Preparing a magnetic core: firstly, 10mg of graphene oxide is dissolved in a solution of 0.5mL of ethylene glycol and 9.5mL of diethylene glycol, and then 0.2830g of sodium acetate and 270mg of ferric chloride are added and mixed uniformly. Then, the mixture was transferred to a stainless steel reactor and heated at 200 ℃ for 10 hours. After the reaction is finished, washing with ethanol to remove unreacted impurities. And transferring the mixed solution into a dialysis bag (MWCO 8000-14000) for slow dialysis for two days. Finally, the product was collected by magnetic separation and resuspended in deionized water.
2) Modification of a chitosan shell: first, 0.1g of chitosan was dissolved in 14mL0.04M acetic acid solution and sufficiently dissolved by ultrasonic dispersion. Then 7mL of the product synthesized in step 1 was taken out, and 1mL0.5M EDC and 1mL0.96M NHS were added at the same time and mixed well. The reaction was carried out for 4 hours under the protection of nitrogen gas, so as to activate the carboxyl groups on the graphene. Subsequently, the chitosan solution prepared above was added and allowed to react for two days at room temperature. After completion of the reaction, the mixture was transferred to a dialysis bag (MWCO 8000-14000) and dialyzed slowly for two days. And finally, collecting the product through magnetic separation, and suspending the product in 50mL of deionized water to obtain the magnetic microsphere.
Example 2
The invention provides a high-efficiency synthesis method of magnetic microspheres, which comprises the following steps:
1) Preparing a magnetic core: firstly, 20mg of graphene oxide is dissolved in a solution of 1mL of ethylene glycol and 19mL of diethylene glycol, then 0.566g of sodium acetate and 540mg of ferric chloride are added, and the mixture is uniformly mixed. Then, the mixture was transferred to a stainless steel reactor and heated at 200 ℃ for 20 hours. After the reaction is finished, washing with ethanol to remove unreacted impurities. And the mixture was transferred to a dialysis bag (MWCO 8000-14000) and dialyzed slowly for two days. Finally, the product was collected by magnetic separation and resuspended in deionized water.
2) Modification of a chitosan shell: firstly, 0.2g of chitosan is dissolved in 28mL0.04M acetic acid solution, and the chitosan is fully dissolved by ultrasonic dispersion. Then, 14mL of the product synthesized in step 1 was taken out, and 2mL0.5M EDC and 2mL0.96M NHS were added at the same time and mixed well. The reaction was carried out for 6 hours under the protection of nitrogen gas, so as to activate the carboxyl groups on the graphene. Subsequently, the chitosan solution prepared above was added and allowed to react for two days at room temperature. After the reaction, the mixture was transferred to a dialysis bag (MWCO 8000-14000) and dialyzed slowly for two days. And finally, collecting the product through magnetic separation, and suspending the product in 100mL of deionized water to obtain the magnetic microsphere.
Example 3
The invention provides a high-efficiency synthesis method of magnetic microspheres, which comprises the following steps:
1) Preparing a magnetic core: firstly, 30mg of graphene oxide is dissolved in a solution of 1.5mL of ethylene glycol and 28.5mL of diethylene glycol, then 0.8490g of sodium acetate and 810mg of ferric chloride are added, and the mixture is uniformly mixed. Then, the mixture was transferred to a stainless steel reactor and heated at 200 ℃ for 30 hours. After the reaction is finished, washing the reaction product by using ethanol to remove unreacted impurities. And transferring the mixed solution into a dialysis bag (MWCO 8000-14000) for slow dialysis for two days. Finally, the product was collected by magnetic separation and resuspended in deionized water.
2) Modification of a chitosan shell: first, 0.3g of chitosan was dissolved in 42mL0.04M acetic acid solution and sufficiently dissolved by ultrasonic dispersion. Then 21mL of the product synthesized in step 1 was taken out, and 3mL0.5M EDC and 3mL0.96M NHS were added at the same time and mixed well. The reaction was carried out for 8 hours under the protection of nitrogen gas, so as to activate the carboxyl groups on the graphene. Subsequently, the chitosan solution prepared above was added and allowed to react for two days at room temperature. After completion of the reaction, the mixture was transferred to a dialysis bag (MWCO 8000-14000) and dialyzed slowly for two days. And finally, collecting the product through magnetic separation, and suspending the product in 150mL of deionized water to obtain the magnetic microsphere.
Example 4
And (3) verifying the effect of purifying and recovering nucleic acid by using magnetic microspheres: nucleic acid recovery Performance test was conducted in the following manner in each of examples 1 to 3, respectively, by Fe hydrothermal synthesis 3 O 4 The nano magnetic microsphere is used as a comparative example 1, the chitosan microsphere prepared by adopting an ion crosslinking method is used as a comparative example 2, and the commercialized AMpureXP magnetic bead is used as a control.
Taking the genome DNA of escherichia coli as a nucleic acid recovery template, preparing the genome DNA template with the total concentration of 100.0 ng/. Mu.L according to a Nano-500 ultramicro spectrophotometer, purifying by using the purified magnetic beads of the invention, and determining the sample recovery rate. Taking 50 mu L of genome DNA template, placing the template in a 1.5ml centrifuge tube, taking 150 mu L of magnetic beads to a sample to be purified, and uniformly mixing the template and the sample to be purified by blowing for not less than 10 times; standing at room temperature for 10min; placing the magnetic bead-sample mixture in the previous step on a magnetic frame and standing for 5min until all the magnetic beads are adsorbed on the magnetic frame; absorbing the supernatant of the last step, adding 300 mu L of 80% ethanol, and standing for 5min; absorbing the supernatant of the last step, adding 300 microliter of prepared 80% ethanol, and standing for 5min; adopting a small-range pipettor to suck and discard the residual alcohol in the previous step again to ensure that no obvious liquid drop alcohol exists; airing the magnetic beads combined with the genome DNA in the previous step at room temperature until no obvious water mark residue exists; DNA elution was carried out with 200. Mu.L of pure water; mixing the eluate with the magnetic beads, and standing at room temperature for 5min; performing instantaneous centrifugation on the magnetic bead eluent in the last step, placing the magnetic bead eluent on a magnetic frame, and standing for 5min until all the magnetic beads are adsorbed by the magnetic frame, wherein the eluent is in a clear and transparent state; absorbing 50 mu L of supernatant clear liquid, and carrying out quantitative detection on the concentration of the genome DNA by using a Nano-500 ultramicro spectrophotometer. The experiment was repeated 3 times per treatment group, and the results were averaged, with no significant difference in 3 data in each treatment group. The measured recovery data are shown in Table 1.
TABLE 1 nucleic acid recovery test data
As can be seen from comparison of the detection data, the average recovery efficiencies of 96.83%, 96.53% and 96.73% for nucleic acids in examples 1 to 3 were comparable to commercially available AMpureXP magnetic beads. Compared with Fe without any modification 3 O 4 For the nano magnetic microsphere, the invention uses oxidized graphene and shellThe modification of the glycan can cause the remarkable improvement of the nucleic acid recovery effect, and the nucleic acid recovery effect is improved from 10% to 96%; compared with chitosan microspheres, the recovery rate of the magnetic microspheres prepared by the method is improved from 30% to 96%, and a remarkable improvement effect is achieved.
Example 5
And (3) verifying the effect of purifying and recovering the protein by using the magnetic microspheres: nucleic acid recovery Performance test was conducted in the following manner in each of examples 1 to 3, respectively, by Fe hydrothermal synthesis 3 O 4 The magnetic nanoparticles were used as comparative example 1, the chitosan microspheres prepared by ion-crosslinking method were used as comparative example 2, and commercial protein-purified magnetic beads (purchased from tokyo-yo-bio-technologies ltd, no. MB 1201) were used as a control.
BSA is used as a protein recovery template, a genome DNA template with the total concentration of 50.0ng/mL is prepared according to a human Bovine Serum Albumin (BSA) ELISA kit (purchased from Shanghai Kanglang Biotechnology Co., ltd., specification 48T/96T, product number KL-BSA-Hu), and the purified magnetic beads are used for purification, so that the sample recovery rate is determined. Taking 50 mu L of total BSA protein template, placing the template in a 1.5ml centrifuge tube, taking 150 mu L of magnetic beads to a sample to be purified, and uniformly pumping the sample for not less than 10 times; standing at room temperature for 10min; placing the magnetic bead-sample mixture in the previous step on a magnetic frame and standing for 5min until all the magnetic beads are adsorbed on the magnetic frame; removing the supernatant, adding 300 μ L of 1 × PBS buffer, and standing for 5min; removing the supernatant, adding 300 μ L of 1 × PBS buffer, and standing for 5min; adopting a small-range pipettor to suck and discard the residual buffer solution in the previous step again to ensure that no obvious liquid drop exists; airing the magnetic beads combined with BSA in the previous step at room temperature until no obvious water mark residue exists; then adding 300 mu L of pure water for elution; mixing the eluate with the magnetic beads, and standing at room temperature for 10min; performing instantaneous centrifugation on the magnetic bead eluent in the last step, placing the magnetic bead eluent on a magnetic frame, and standing for 10min until all the magnetic beads are adsorbed by the magnetic frame, wherein the eluent is in a clear and transparent state; 50 μ L of supernatant was aspirated, and the total protein concentration was quantitatively determined using human Bovine Serum Albumin (BSA) ELISA kit. The experiment was repeated 3 times per treatment group, and the results were averaged, with no significant difference in 3 data in each treatment group. The measured recovery data are shown in Table 2.
TABLE 2 protein recovery test data
As is clear from comparison of the detection data, the average recovery efficiencies of 92.46%, 92.36% and 92.56% for the BSA proteins in examples 1 to 3 were comparable to those of commercially available protein-purified magnetic beads. Compared with Fe without any modification 3 O 4 For the nano magnetic microspheres, the protein recovery effect is remarkably improved from 8% to 92% after the nano magnetic microspheres are modified by graphene oxide and chitosan; compared with chitosan microspheres, the recovery rate of the magnetic microspheres prepared by the method is improved from 20% to 92%, and a significant improvement effect is achieved.
Therefore, it should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A high-efficiency synthesis method of magnetic microspheres for separating and purifying nucleic acid and/or protein is characterized by comprising the following steps:
1) Preparing a magnetic core: preparing graphene oxide and ferric chloride by a hydrothermal synthesis method to obtain a magnetic core;
2) Modification of a chitosan shell: modifying a chitosan shell on the surface of the magnetic core synthesized in the step 1) to prepare the magnetic microsphere.
2. The method of synthesis according to claim 1, wherein the method of synthesis of step 1) comprises the steps of: firstly, dissolving graphene oxide in a solution of ethylene glycol and diethylene glycol, then adding sodium acetate and ferric chloride, and uniformly mixing; then, transferring the mixed solution into a stainless steel reaction kettle, and heating at 200 ℃; after the reaction is finished, cleaning the mixture by using ethanol to remove unreacted impurities; transferring the mixed solution into a dialysis bag, and slowly dialyzing for two days; finally, the product was collected by magnetic separation and resuspended in deionized water.
3. The synthesis method according to claim 2, wherein the synthesis method of step 1) comprises the steps of: firstly, dissolving 10-30mg of graphene oxide in a solution of 0.5-1.5mL of ethylene glycol and 9.5-28.5mL of diethylene glycol, then adding 0.2830-0.8490g of sodium acetate and 270-810mg of ferric chloride, and uniformly mixing; then, transferring the mixed solution into a stainless steel reaction kettle, and heating for 10-30 hours at the temperature of 200 ℃; after the reaction is finished, cleaning the mixture by using ethanol to remove unreacted impurities; transferring the mixed solution into a dialysis bag (MWCO 8000-14000), and slowly dialyzing for two days; finally, the product was collected by magnetic separation and resuspended in deionized water.
4. The method of synthesis according to claim 1, wherein the method of synthesis of step 2) comprises the steps of: firstly, dissolving chitosan in an acetic acid solution, and fully dissolving the chitosan through ultrasonic dispersion; then taking out the product synthesized in the step 1), adding EDC and NHS at the same time, and mixing uniformly; under the protection of nitrogen, carrying out reaction so as to activate carboxyl on the graphene; then, adding the prepared chitosan solution, and reacting for two days at room temperature; after the reaction is finished, similarly transferring the mixed solution into a dialysis bag, and slowly dialyzing for two days; and finally, collecting the product through magnetic separation, and suspending the product in deionized water to obtain the magnetic microsphere.
5. The method of synthesis according to claim 4, wherein the method of synthesis of step 2) comprises the steps of: firstly, 0.1-0.3g of chitosan is dissolved in 14-42mL0.04M acetic acid solution, and the chitosan is fully dissolved through ultrasonic dispersion; then taking out 7-21mL of the product synthesized in the step 1), simultaneously adding 1-3mL0.5M EDC and 1-3mL0.96M NHS, and uniformly mixing; reacting for 4-8 hours under the protection of nitrogen so as to activate carboxyl on the graphene; then, adding the prepared chitosan solution, and reacting for two days at room temperature; after the reaction is finished, the mixed solution is transferred into a dialysis bag (MWCO 8000-14000) and slowly dialyzed for two days; and finally, collecting the product through magnetic separation, and suspending the product in 50-150mL of deionized water to obtain the magnetic microsphere.
6. Use of magnetic microspheres prepared by the method of any one of claims 1-5 for the separation and purification of nucleic acids and/or proteins.
7. The use of claim 6, wherein the nucleic acid separation and purification can be achieved by contacting the magnetic microspheres with a nucleic acid sample to be separated and performing magnetic separation.
8. The use of claim 6, wherein the protein is isolated and purified by contacting the magnetic microspheres with a protein sample to be isolated and performing magnetic separation.
9. The use according to claim 7, wherein the nucleic acid sample to be isolated is the genomic DNA of E.coli.
10. The use according to claim 8 wherein the protein sample to be separated is BSA.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105241860A (en) * | 2015-11-20 | 2016-01-13 | 济南大学 | Method for preparing magnetic fluorescent graphene composite nano ion probes |
| CN107137707A (en) * | 2017-04-01 | 2017-09-08 | 国家纳米科学中心 | Chitosan-modified magnetic heterojunction structure and its application at antibacterial aspect |
| CN109735532A (en) * | 2018-12-30 | 2019-05-10 | 北京优迅医学检验实验室有限公司 | A kind of bead suspension and its application for nucleic acid purification and screening |
| CN110233020A (en) * | 2019-06-14 | 2019-09-13 | 迪佰(厦门)生物科技有限公司 | A kind of extracting method for the magnetic material of nucleic acid extraction, nucleic acid-extracting apparatus and nucleic acid |
| CN111330558A (en) * | 2020-03-10 | 2020-06-26 | 上海领骏生物科技有限公司 | Method for manufacturing magnetic microspheres for extracting and purifying trace nucleic acid |
| CN113293159A (en) * | 2021-06-21 | 2021-08-24 | 清华大学深圳国际研究生院 | Nucleic acid extraction kit and nucleic acid extraction method |
-
2023
- 2023-02-15 CN CN202310113091.9A patent/CN115970654A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105241860A (en) * | 2015-11-20 | 2016-01-13 | 济南大学 | Method for preparing magnetic fluorescent graphene composite nano ion probes |
| CN107137707A (en) * | 2017-04-01 | 2017-09-08 | 国家纳米科学中心 | Chitosan-modified magnetic heterojunction structure and its application at antibacterial aspect |
| CN109735532A (en) * | 2018-12-30 | 2019-05-10 | 北京优迅医学检验实验室有限公司 | A kind of bead suspension and its application for nucleic acid purification and screening |
| CN110233020A (en) * | 2019-06-14 | 2019-09-13 | 迪佰(厦门)生物科技有限公司 | A kind of extracting method for the magnetic material of nucleic acid extraction, nucleic acid-extracting apparatus and nucleic acid |
| CN111330558A (en) * | 2020-03-10 | 2020-06-26 | 上海领骏生物科技有限公司 | Method for manufacturing magnetic microspheres for extracting and purifying trace nucleic acid |
| CN113293159A (en) * | 2021-06-21 | 2021-08-24 | 清华大学深圳国际研究生院 | Nucleic acid extraction kit and nucleic acid extraction method |
Non-Patent Citations (1)
| Title |
|---|
| NENGSHENG YE ET AL.: "Synthesis of magnetite/graphene oxide/chitosan composite and its application for protein adsorption", 《MATERIALS SCIENCE AND ENGINEERING C》, vol. 45, pages 2 - 2 * |
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